Recently, a large number of measurements using intermolecular interactions such as immune responses are being carried out in clinical tests, etc. However, since conventional methods require complicated operations or labeling substances, several techniques are used that are capable of detecting the change in the binding amount of a test substance with high sensitivity without using such labeling substances. Examples of such a technique may include a surface plasmon resonance (SPR) measurement technique, a quartz crystal microbalance (QCM) measurement technique, and a measurement technique of using functional surfaces ranging from gold colloid particles to ultra-fine particles. The SPR measurement technique is a method of measuring changes in the refractive index near an organic functional film attached to the metal film of a chip by measuring a peak shift in the wavelength of reflected light, or changes in amounts of reflected light in a certain wavelength, so as to detect adsorption and desorption occurring near the surface. The QCM measurement technique is a technique of detecting adsorbed or desorbed mass at the ng level, using a change in frequency of a crystal due to adsorption or desorption of a substance on gold electrodes of a quartz crystal (device). In addition, the ultra-fine particle surface (nm level) of gold is functionalized, and physiologically active substances are immobilized thereon. Thus, a reaction to recognize specificity among physiologically active substances is carried out, thereby detecting a substance associated with a living organism from sedimentation of gold fine particles or sequences.
In all of the above-described techniques, the surface where a physiologically active substance is immobilized is important. Surface plasmon resonance (SPR), which is most commonly used in this technical field, will be described below as an example.
A commonly used measurement chip comprises a transparent substrate (e.g., glass), an evaporated metal film, and a thin film having thereon a functional group capable of immobilizing a physiologically active substance. The measurement chip immobilizes the physiologically active substance on the metal surface via the functional group. A specific binding reaction between the physiological active substance and a test substance is measured, so as to analyze an interaction between biomolecules.
Japanese Patent No. 2815120 discloses in detail a method for producing hydrogel, which is used as a detection surface having a functional group capable of immobilizing a physiologically active substance, for example. Specifically, a 16-mercaptohexadecanol layer binds to a gold film, so as to form a barrier layer. On the gold film, a hydroxyl group of the barrier layer is treated with epichlorohydrin so as to be epoxy-activated. In the subsequent step, dextran is allowed to bind to the barrier layer via an ether bond. Then, bromoacetic acid is allowed to react with a dextran matrix, resulting in introduction of a carboxymethyl group.
As a method for immobilizing a physiologically active substance having an amino group (e.g. a protein or amino acid) on the carboxymethyl degeneration dextran surface, which is produced by the above method, the following method has been disclosed. That is to say, a portion of carboxyl groups in carboxymethyl degeneration dextran are treated with an aqueous solution that contains N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) hydrochloride, for example, so that such groups are degenerated so as to achieve a reactive ester function. Residual charge, that is, unreacted carboxyl groups would contribute to concentration of a physiologically active substance onto a detection surface. An aqueous solution of a physiologically active substance containing an amino group (a protein or amino acid) is allowed to come into contact with such a detection surface, so as to allow the physiologically active substance containing an amino group to bind to a dextran matrix via a covalent bond.
Since such hydrogel produced by the aforementioned method is able to immobilize a physiologically active substance containing an amino group in a three-dimensional manner, it has outstanding performance as a detection surface of biosensors. However, the aforementioned production method of hydrogel is complicated, and it requires a long production time. Further, since the above production method requires the use of compounds such as epichlorohydrin or bromoacetic acid, it has been problematic in terms of safety.
On the other hand, with regard to immobilization of a substance utilizing a photoreactive group, introduction of a photoreactive group (an azidophenyl group, etc.) into a polymer, so as to allow the polymer together with a protein to bind to a substrate (JP Patent Publication (Kokai) No. 2004-125781A), introduction of a photoreactive group (benzophenone) into a substrate, so as to allow a hydrophobic polymer (polystyrene, polyethyloxazolidine) to bind to the substrate (J. Am. Chem. Soc., 121, 8766 (1999)), immobilization of a DNA fragment on a solid surface via a graft polymer, so as to produce a DNA chip (JP Patent Publication (Kokai) No. 2003-130878A), and the like, have been reported.